Continuous monitoring of normal uterine contractile activity during late term pregnancy in humans has shown increased frequency between the hours of 8:30 PM and 2:00 AM1. Studies on the timing of human labor onset and deliveries show that the initiation of labor peaks between the hours of 24:00 and 05:00, regardless of gestational age2. Current models describe parturition as a multi-step process beginning with myometrial activation in late pregnancy followed by stimulation leading to uterine contraction and subsequent delivery of the infant. Myometrial activation encompasses cellular remodeling with appropriate changes in gene expression. The increased expression of these “contraction-associated proteins” marks the transition of the myometrium from a quiescent to activated state. These proteins facilitate the powerful uterine contractions necessary to deliver the infant by increasing the excitability of the myometrial cells, enhancing smooth muscle myosin-actin interactions, and increasing intercellular connectivity, thereby facilitating synchronous myometrial contractions3.
After its activation the myometrium can be stimulated by multiple factors including oxytocin, prostaglandins and noradrenaline4, 5. Oxytocin, a nonapeptide hormone secreted by the pituitary gland is one of the most potent uterine contractants. Oxytocin (OT), upon binding to its Gq-protein coupled receptor (OTR), activates the membrane-bound phospholipase C (PLC) and subsequently protein kinase C (PKC). Inositol trisphosphate (IP3), cleaved from membrane phospholipids, binds receptors on the sarcoplasmic reticulum triggering the release of Ca++ from intracellular stores as well as the influx of extracellular Ca++ via membrane calcium channels. Increases in intracellular calcium concentrations result in activation of the Ca++/calmodulindependent enzyme, myosin light chain kinase (MLCK), thereby leading to the phosphorylation of the myosin light chain and also to myometrial contraction6, 7.
Melatonin (MEL), a monoamine hormone secreted by the epithalamic pineal gland, is a major molecular messenger of the nocturnal phase of the light-dark cycle. MEL signals via two G-protein coupled receptors, melatonin receptor 1 (MT1R) and melatonin receptor 2 (MT2R)8. We have previously shown that human myometrium is a target for MEL and expresses both melatonin receptors9, 10. These studies pointed to a potential point of interaction between OT and MEL signaling pathways. An earlier report demonstrated that MEL potentiates norepinephrine-induced contractility in a dose dependent manner in human myometrial strips11.
Our previous work showed striking similarities between MEL regulation of OTR mRNA expression and the regulation of OTR mRNA expression by OT10, 12 leading us to further explore the similarities between the MEL and OT signaling pathways in the myometrium. Herein, we investigated the effects of MEL on myometrial contractility in vitro by conducting experiments with the well characterized hTERT telomerase-immortalized human myometrial smooth muscle cell line which have been shown to express oxytocin receptors13. The results of the present studies show that MEL acts synergistically via the MT2R/PLC/PKC signaling pathway to significantly increase sensitivity of myocytes to OT and increase OT-induced contractility in a dose dependent manner. In addition, we also investigated the potential effects of MEL on expression of the gap junction protein, connexin43 (Cx43). Expression of Cx43 is known to increase late in human pregnancy thereby facilitating myometrial cell coupling and synchronization of uterine contraction3. Our data reveal that MEL treatment of cultured myometrial cells increased both mRNA and protein levels of Cx43 via the MT2R signaling cascade. Taken together these studies point to a novel regulatory function of the circadian hormone MEL in “gating” human myometrial activity. More specifically, our data provide a model system to investigate the mechanism through which MEL interacts with the OT pathway to promote uterine contractility and parturition.